2,5-diketo-D-gluconic acid reductase (2,5-DKGR; E.C. 1.1.1.-) catalyzes the Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent stereo-specific reduction of 2, 5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate (2-KLG), a precursor in the industrial production of vitamin C (L-ascorbate). Microorganisms that naturally ferment D-glucose to 2,5-DKG can be genetically modified to express the gene for 2,5-DKGR, and thus directly produce vitamin C from D-glucose. Two naturally occurring... Show more2,5-diketo-D-gluconic acid reductase (2,5-DKGR; E.C. 1.1.1.-) catalyzes the Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent stereo-specific reduction of 2, 5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate (2-KLG), a precursor in the industrial production of vitamin C (L-ascorbate). Microorganisms that naturally ferment D-glucose to 2,5-DKG can be genetically modified to express the gene for 2,5-DKGR, and thus directly produce vitamin C from D-glucose. Two naturally occurring variants of DKGR (DKGR A and DKGR B) have been reported. DKGR B exhibits higher specific activity toward 2,5-DKG than DKGR A; however, DKGR A exhibits a greater selectivity for this substrate and significantly higher thermal stability. Thus, a modified form of DKGR, combining desirable properties from both enzymes, would be of substantial commercial interest. In the present study we use a molecular dynamics-based approach to understand the conformational changes in DKGR A as the active site is mutated to include two active site residue changes that occur in the B form. The results indicate that the enhanced kinetic properties of the B form are due, in part, to residue substitutions in the binding pocket. These substitutions augment interactions with the substrate or alter the alignment with respect to the putative proton donor group. Show less

The human kallikreins are a large multigene family of closely related serine-type proteases. In this regard, they are similar to the multigene kallikrein families characterized in mice and rats. There is a much more extensive body of knowledge regarding the function of mouse and rat kallikreins in comparison with the human kallikreins. Human kallikrein 6 has been proposed as the homologue to rat myelencephalon-specific protease, an arginine-specific degradative-type protease abundantly... Show moreThe human kallikreins are a large multigene family of closely related serine-type proteases. In this regard, they are similar to the multigene kallikrein families characterized in mice and rats. There is a much more extensive body of knowledge regarding the function of mouse and rat kallikreins in comparison with the human kallikreins. Human kallikrein 6 has been proposed as the homologue to rat myelencephalon-specific protease, an arginine-specific degradative-type protease abundantly expressed in the central nervous system and implicated in demyelinating disease. We present the x-ray crystal structure of mature, active recombinant human kallikrein 6 at 1.75-Å resolution. This high resolution model provides the first three-dimensional view of one of the human kallikreins and one of only a few structures of serine proteases predominantly expressed in the central nervous system. Enzymatic data are presented that support the identification of human kallikrein 6 as the functional homologue of rat myelencephalon-specific protease and are corroborated by a molecular phylogenetic analysis. Furthermore, the x-ray data provide support for the characterization of human kallikrein 6 as a degradative protease with structural features more similar to trypsin than the regulatory kallikreins. Show less

Background Our aim was to determine if pramipexole, a D3 preferring agonist, effectively reduced dopamine neuron and fiber loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model when given at intraperitoneal doses corresponding to clinical doses. We also determined whether subchronic treatment with pramipexole regulates dopamine transporter function, thereby reducing intracellular transport of the active metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPP+). Methods Ten... Show moreBackground Our aim was to determine if pramipexole, a D3 preferring agonist, effectively reduced dopamine neuron and fiber loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model when given at intraperitoneal doses corresponding to clinical doses. We also determined whether subchronic treatment with pramipexole regulates dopamine transporter function, thereby reducing intracellular transport of the active metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPP+). Methods Ten 12-month old C57BL/6 mice were treated with MPTP (or saline) twice per day at 20 mg/kg s.c. (4 injections over 48 h). Mice were pretreated for 3 days and during the 2-day MPTP regimen with pramipexole (0.1 mg/kg/day) or saline. Stereological quantification of dopamine neuron number and optical density measurement of dopamine fiber loss were carried out at 1 week after treatment, using immunostaining for dopamine transporter (DAT) and tyrosine hydroxylase (TH). Additional wild-type (WT) and D3 receptor knockout (KO) mice were treated for 5 days with pramipexole (0.1 mg/kg/day) or vehicle. The kinetics of [3H]MPP+ and [3H]DA uptake (V max and K m) were determined 24 h later; and at 24 h and 14 days dopamine transporter density was measured by quantitative autoradiography. Results Pramipexole treatment completely antagonized the neurotoxic effects of MPTP, as measured by substantia nigra and ventral tegmental area TH-immunoreactive cell counts. MPTP- induced loss of striatal innervation, as measured by DAT-immunoreactivity, was partially prevented by pramipexole, but not with regard to TH-IR. Pramipexole also reduced DAT- immunoreactivity in non-MPTP treated mice. Subchronic treatment with pramipexole lowered the V max for [3H]DA and [3H]MPP+ uptake into striatal synaptosomes of WT mice. Pramipexole treatment lowered V max in WT but not D3 KO mice; however, D3 KO mice had lower V max for [3H]DA uptake. There was no change in DAT number in WT with pramipexole treatment or D3 KO mice at 24 h post-treatment, but there was a reduction in WT-pramipexole treated and not in D3 KO mice at 14 days post-treatment. Conclusion These results suggest that protection occurs at clinically suitable doses of pramipexole. Protection could be due to a reduced amount of MPP+ taken up into DA terminals via DAT. D3 receptor plays an important role in this regulation of transporter uptake and availability. Show less

In budding yeast Saccharomyces cerevisiae, Cdc5 kinase is a component of mitotic exit network (MEN), which inactivates cyclin-dependent kinase (CDK) after chromosome segregation. cdc5-1 mutants arrest at telophase at the nonpermissive temperature due to the failure of CDK inactivation. To identify more negative regulators of MEN, we carried out a genetic screen for genes that are toxic to cdc5-1 mutants when overexpressed. Genes that encode the B-regulatory subunit (Cdc55) and the three... Show moreIn budding yeast Saccharomyces cerevisiae, Cdc5 kinase is a component of mitotic exit network (MEN), which inactivates cyclin-dependent kinase (CDK) after chromosome segregation. cdc5-1 mutants arrest at telophase at the nonpermissive temperature due to the failure of CDK inactivation. To identify more negative regulators of MEN, we carried out a genetic screen for genes that are toxic to cdc5-1 mutants when overexpressed. Genes that encode the B-regulatory subunit (Cdc55) and the three catalytic subunits (Pph21, Pph22, and Pph3) of phosphatase 2A (PP2A) were isolated. In addition to cdc5-1, overexpression of CDC55, PPH21, or PPH22 is also toxic to other temperature-sensitive mutants that display defects in mitotic exit. Consistently, deletion of CDC55 partially suppresses the temperature sensitivity of these mutants. Moreover, in the presence of spindle damage, PP2A mutants display nuclear localized Cdc14, the key player in MEN pathway, indicative of MEN activation. All the evidence suggests the negative role of PP2A in mitotic exit. Finally, our genetic and biochemical data suggest that PP2A regulates the phosphorylation of Tem1, which acts at the very top of MEN pathway. Show less

The function and regulation of budding yeast Swe1 in response to interrupted DNA synthesis.

Creator

Liu, Hong, Wang, Yanchang

Abstract/Description

Periodically regulated cyclin-dependent kinase (Cdk) is required for DNA synthesis and mitosis. Hydroxyurea (HU) inhibits DNA synthesis by depleting dNTPs, the basic unit for DNA synthesis. HU treatment triggers the S-phase checkpoint, which arrests cells at S-phase, inhibits late origin firing and stabilizes replication forks. Using budding yeast as a model system, we found that Swe1, a negative regulator of Cdk, appears at S-phase and accumulates in HU treatment cells. Interestingly, this... Show morePeriodically regulated cyclin-dependent kinase (Cdk) is required for DNA synthesis and mitosis. Hydroxyurea (HU) inhibits DNA synthesis by depleting dNTPs, the basic unit for DNA synthesis. HU treatment triggers the S-phase checkpoint, which arrests cells at S-phase, inhibits late origin firing and stabilizes replication forks. Using budding yeast as a model system, we found that Swe1, a negative regulator of Cdk, appears at S-phase and accumulates in HU treatment cells. Interestingly, this accumulation is not dependent on S-phase checkpoint. Deltahsl1, Deltahsl7, and cdc5-2 mutants, which have defects in Swe1 degradation, show HU sensitivity because of high Swe1 protein levels. We further demonstrated that their HU sensitivity is not a result of DNA damage accumulation or incomplete DNA synthesis; instead the sensitivity is due to their dramatically delayed recovery from HU-induced S-phase arrest. Strikingly, our in vivo data indicate that Swe1 inhibits the kinase activity of Clb2-Cdk1, but not that of Clb5-Cdk1. Therefore, S-phase accumulated Swe1 prevents Clb2-Cdk1-mediated mitotic activities, but has little effects on Clb5-Cdk1-associated S-phase progression. Show less

Spindle disruption or DNA damage prevents sister chromatid separation through the activation of checkpoint pathways that inhibit anaphase entry by stabilizing the anaphase inhibitor Pds1. Mutation of CDC55, which encodes a B regulatory subunit of protein phosphatase 2A (PP2A), results in precocious sister chromatid separation when spindle is disrupted. Here we report that decreased Pds1 levels in Deltacdc55 mutants contribute to sister chromatid separation in the presence of nocodazole, a... Show moreSpindle disruption or DNA damage prevents sister chromatid separation through the activation of checkpoint pathways that inhibit anaphase entry by stabilizing the anaphase inhibitor Pds1. Mutation of CDC55, which encodes a B regulatory subunit of protein phosphatase 2A (PP2A), results in precocious sister chromatid separation when spindle is disrupted. Here we report that decreased Pds1 levels in Deltacdc55 mutants contribute to sister chromatid separation in the presence of nocodazole, a microtubule-depolymerizing drug. However, in the presence of DNA damage, Deltacdc55 mutant cells separate sister chromatids without noticeable decrease of Pds1 or cohesin Mcd1/Scc1 levels. Further analysis demonstrates that Deltacdc55 mutants lose cohesion along the entire chromosomes when the spindle is disrupted. In contrast, separation of sister chromatids is limited to the centromeric regions in Deltacdc55 cells after DNA damage. Moreover, mutation of TPD3, which encodes the A regulatory subunit of PP2A, also results in sister chromatid separation in DNA- or spindle-damage-arrested cells. These data suggest that PP2A regulates sister chromatid cohesion in Pds1-dependent and -independent manners. Show less

The in vivo imaging probe [11C]-PIB (Pittsburgh Compound B, N-methyl[11C]2-(4'-methylaminophenyl-6-hydroxybenzathiazole) is under evaluation as a key imaging tool in Alzheimer's disease (AD) and to date has been assumed to bind with high affinity and specificity to the amyloid structures associated with classical plaques (CPs), one of the pathological hallmarks of the disease. However, no studies have systematically investigated PIB binding to human neuropathological brain specimens at the... Show moreThe in vivo imaging probe [11C]-PIB (Pittsburgh Compound B, N-methyl[11C]2-(4'-methylaminophenyl-6-hydroxybenzathiazole) is under evaluation as a key imaging tool in Alzheimer's disease (AD) and to date has been assumed to bind with high affinity and specificity to the amyloid structures associated with classical plaques (CPs), one of the pathological hallmarks of the disease. However, no studies have systematically investigated PIB binding to human neuropathological brain specimens at the tracer concentrations achieved during in vivo imaging scans. Using a combination of autoradiography and histochemical techniques, we demonstrate that PIB, in addition to binding CPs clearly delineates diffuse plaques and cerebrovascular amyloid angiopathy (CAA). The interaction of PIB with CAA was not fully displaceable and this may be linked to the apolipoprotein E-epsilon4 allele. PIB was also found to label neurofibrillary tangles, although the overall intensity of this binding was markedly lower than that associated with the amyloid-beta (Abeta) pathology. The data provide a molecular explanation for PIB's limited specificity in diagnosing and monitoring disease progression in AD and instead indicate that the ligand is primarily a non-specific marker of Abeta-peptide related cerebral amyloidosis. Show less

Human kallikrein-related peptidase 6 (KLK6) is a member of the kallikrein family of serine-type proteases, characterized as an arginine-specific digestive-type protease capable of degrading a wide-variety of extracellular matrix proteins. KLK6 has been proposed to be a useful biomarker for breast and ovarian cancer prognosis, is abundantly expressed in the CNS and cerebrospinal fluid, and is intimately associated with regions of active inflammatory demyelination in multiple sclerosis (MS)... Show moreHuman kallikrein-related peptidase 6 (KLK6) is a member of the kallikrein family of serine-type proteases, characterized as an arginine-specific digestive-type protease capable of degrading a wide-variety of extracellular matrix proteins. KLK6 has been proposed to be a useful biomarker for breast and ovarian cancer prognosis, is abundantly expressed in the CNS and cerebrospinal fluid, and is intimately associated with regions of active inflammatory demyelination in multiple sclerosis (MS) lesions. Inhibition of KLK6 results in delayed onset and reduced severity of symptoms associated with experimental autoimmune encephalomyelitis, suggesting a key effector role for this protease in CNS inflammatory disease. KLK6 has been shown to autolytically cleave internally, leading to inactivation and suggesting a negative feedback inhibition control mechanism. Alternatively, the ability of KLK6 to self-activate has also been reported, suggesting a positive feedback activation loop control mechanism. Activation of pro-KLK6 requires hydrolysis after a Lys residue; however, KLK6 exhibits 2 order of magnitude reduced affinity for hydrolysis after Lys versus Arg residues; therefore, the ability to autolytically activate has been called into question. In the present study the catalytic activity of KLK6 toward its pro-sequence and internal autolytic sequence is characterized. The results show that the ability of KLK6 to activate pro-KLK6 is essentially negligible when compared to the rate of the internal autolytic inactivation or to the ability of other proteases to activate pro-KLK6. The results thus show that the primary autolytic regulatory mechanism of KLK6 is negative feedback inhibition, and activation is likely achieved through the action of a separate protease. Show less

DNA damage checkpoints inhibit mitotic exit by two different mechanisms.

Creator

Liang, Fengshan, Wang, Yanchang

Abstract/Description

Cyclin-dependent kinase (CDK) governs cell cycle progression, and its kinase activity fluctuates during the cell cycle. Mitotic exit pathways are responsible for the inactivation of CDK after chromosome segregation by promoting the release of a nucleolus-sequestered phosphatase, Cdc14, which antagonizes CDK. In the budding yeast Saccharomyces cerevisiae, mitotic exit is controlled by the FEAR (for "Cdc-fourteen early anaphase release") and mitotic exit network (MEN) pathways. In response to... Show moreCyclin-dependent kinase (CDK) governs cell cycle progression, and its kinase activity fluctuates during the cell cycle. Mitotic exit pathways are responsible for the inactivation of CDK after chromosome segregation by promoting the release of a nucleolus-sequestered phosphatase, Cdc14, which antagonizes CDK. In the budding yeast Saccharomyces cerevisiae, mitotic exit is controlled by the FEAR (for "Cdc-fourteen early anaphase release") and mitotic exit network (MEN) pathways. In response to DNA damage, two branches of the DNA damage checkpoint, Chk1 and Rad53, are activated in budding yeast to prevent anaphase entry and mitotic exit, allowing cells more time to repair damaged DNA. Here we present evidence indicating that yeast cells negatively regulate mitotic exit through two distinct pathways in response to DNA damage. Rad53 prevents mitotic exit by inhibiting the MEN pathway, whereas the Chk1 pathway prevents FEAR pathway-dependent Cdc14 release in the presence of DNA damage. In contrast to previous data, the Rad53 pathway negatively regulates MEN independently of Cdc5, a Polo-like kinase essential for mitotic exit. Instead, a defective Rad53 pathway alleviates the inhibition of MEN by Bfa1. Show less

Effects of chronic environmental and social stimuli during adolescence on mesolimbic dopaminergic circuitry markers.

Creator

Kabbaj, M, Isgor, C

Abstract/Description

Previously, we have shown that chronic exposure to environmental and social stimuli (ESS) during adolescence prevents the development of behavioral sensitization to amphetamine in adult rats. At the onset of the peripubertal-juvenile period (28-d) male rats were subjected to a 28-d long intermittent ESS protocol or handled as controls (NO-ESS). Twenty-four hours after the last session of ESS or NO-ESS, all rats started a regimen of behavioral sensitization to amphetamine (1mg/kg, i.p.), in... Show morePreviously, we have shown that chronic exposure to environmental and social stimuli (ESS) during adolescence prevents the development of behavioral sensitization to amphetamine in adult rats. At the onset of the peripubertal-juvenile period (28-d) male rats were subjected to a 28-d long intermittent ESS protocol or handled as controls (NO-ESS). Twenty-four hours after the last session of ESS or NO-ESS, all rats started a regimen of behavioral sensitization to amphetamine (1mg/kg, i.p.), in which rats were injected every third day with amphetamine or saline on four occasions. Then following one week abstinence all rats were challenged with a lower dose of amphetamine (0.5mg/kg, i.p.) and their locomotor activity monitored for 2h. Our results showed that while NO-ESS rats developed behavioral sensitization to amphetamine, ESS rats did not develop this behavior. All rats were then sacrificed 3 days following the challenge to allow for amphetamine clearance. Since mesolimbic dopamine has been implicated in behavioral sensitization to amphetamine we compared messenger RNA (mRNA) expression of key dopamine-related molecules in the mesolimbic circuitry in ESS and NO-ESS rats. A decrease in dopaminergic D1 receptor (D1R) gene expression in the caudate-putamen (CPu) was associated with amphetamine sensitization in the controls, possibly as a result of a chronic increase in DA release. In contrast, amphetamine treatment did not modulate D1R mRNA levels in ESS rats. No change has been detected in any other dopaminergic markers [D2R, D3R, tyrosine hydroxylase (TH) or dopamine transporter (DAT) mRNAs]. Consequently, we conclude that ESS may inhibit the development of behavioral sensitization to amphetamine through preventing the decrease in CPu D1R mRNA levels. Show less

The human kallikrein-related peptidases (KLKs) comprise 15 members (KLK1-15) and are the single largest family of serine proteases. The KLKs are utilized, or proposed, as clinically important biomarkers and therapeutic targets of interest in cancer and neurodegenerative disease. All KLKs appear to be secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their N-terminal pro-peptide. This processing is a key step in the regulation of... Show moreThe human kallikrein-related peptidases (KLKs) comprise 15 members (KLK1-15) and are the single largest family of serine proteases. The KLKs are utilized, or proposed, as clinically important biomarkers and therapeutic targets of interest in cancer and neurodegenerative disease. All KLKs appear to be secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their N-terminal pro-peptide. This processing is a key step in the regulation of KLK function. Much recent work has been devoted to elucidating the potential for activation cascades between members of the KLK family, with physiologically relevant KLK regulatory cascades now described in skin desquamation and semen liquefaction. Despite this expanding knowledge of KLK regulation, details regarding the potential for functional intersection of KLKs with other regulatory proteases are essentially unknown. To elucidate such interaction potential, we have characterized the ability of proteases associated with thrombostasis to hydrolyze the pro-peptide sequences of the KLK family using a previously described pro-KLK fusion protein system. A subset of positive hydrolysis results were subsequently quantified with proteolytic assays using intact recombinant pro-KLK proteins. Pro-KLK6 and 14 can be activated by both plasmin and uPA, with plasmin being the best activator of pro-KLK6 identified to date. Pro-KLK11 and 12 can be activated by a broad-spectrum of thrombostasis proteases, with thrombin exhibiting a high degree of selectivity for pro-KLK12. The results show that proteases of the thrombostasis family can efficiently activate specific pro-KLKs, demonstrating the potential for important regulatory interactions between these two major protease families. Show less

Tissue kallikrein KLK1 and the kallikrein-related peptidases KLK2-15 are a subfamily of serine proteases that have defined or proposed roles in a range of central nervous system (CNS) and non-CNS pathologies. To further understand their potential activity in multiple sclerosis (MS), serum levels of KLK1, 6, 7, 8 and 10 were determined in 35 MS patients and 62 controls by quantitative fluorometric ELISA. Serum levels were then correlated with Expanded Disability Status Scale (EDSS) scores... Show moreTissue kallikrein KLK1 and the kallikrein-related peptidases KLK2-15 are a subfamily of serine proteases that have defined or proposed roles in a range of central nervous system (CNS) and non-CNS pathologies. To further understand their potential activity in multiple sclerosis (MS), serum levels of KLK1, 6, 7, 8 and 10 were determined in 35 MS patients and 62 controls by quantitative fluorometric ELISA. Serum levels were then correlated with Expanded Disability Status Scale (EDSS) scores determined at the time of serological sampling or at last clinical follow-up. Serum levels of KLK1 and KLK6 were elevated in MS patients (p Show less

Obtaining synchronous cell populations is essential for cell-cycle studies. Methods such as serum withdrawal or use of drugs which block cells at specific points in the cell cycle alter cellular events upon re-entry into the cell cycle. Regulatory events occurring in early G1 phase of a new cell cycle could have been overlooked. We used a robotic mitotic shake-off apparatus to select cells in late mitosis for genome-wide gene expression studies. Two separate microarray experiments were... Show moreObtaining synchronous cell populations is essential for cell-cycle studies. Methods such as serum withdrawal or use of drugs which block cells at specific points in the cell cycle alter cellular events upon re-entry into the cell cycle. Regulatory events occurring in early G1 phase of a new cell cycle could have been overlooked. We used a robotic mitotic shake-off apparatus to select cells in late mitosis for genome-wide gene expression studies. Two separate microarray experiments were conducted, one which involved isolation of RNA hourly for several hours from synchronous cell populations, and one experiment which examined gene activity every 15 minutes from late telophase of mitosis into G1 phase. To verify synchrony of the cell populations under study, we utilized methods including BrdU uptake, FACS, and microarray analyses of histone gene activity. We also examined stress response gene activity. Our analysis enabled identification of 200 early G1-regulated genes, many of which currently have unknown functions. We also confirmed the expression of a set of genes candidates (fos, atf3 and tceb) by qPCR to further validate the newly identified genes. Genome-scale expression analyses of the first two hours of G1 in naturally cycling cells enabled the discovery of a unique set of G1-regulated genes, many of which currently have unknown functions, in cells progressing normally through the cell division cycle. This group of genes may contain future targets for drug development and treatment of human disease. Show less

Hypertrophic Cardiomyopathy (HCM) is a common primary cardiac disorder defined by a hypertrophied left ventricle, is one of the main causes of sudden death in young athletes, and has been associated with mutations in most sarcomeric proteins (tropomyosin, troponin T and I, and actin, etc.). Many of these mutations appear to affect the functional properties of cardiac troponin C (cTnC), i.e., by increasing the Ca(2+)-sensitivity of contraction, a hallmark of HCM, yet surprisingly, prior to... Show moreHypertrophic Cardiomyopathy (HCM) is a common primary cardiac disorder defined by a hypertrophied left ventricle, is one of the main causes of sudden death in young athletes, and has been associated with mutations in most sarcomeric proteins (tropomyosin, troponin T and I, and actin, etc.). Many of these mutations appear to affect the functional properties of cardiac troponin C (cTnC), i.e., by increasing the Ca(2+)-sensitivity of contraction, a hallmark of HCM, yet surprisingly, prior to this report, cTnC had not been classified as a HCM-susceptibility gene. In this study, we show that mutations occurring in the human cTnC (HcTnC) gene (TNNC1) have the same prevalence (~0.4%) as well established HCM-susceptibility genes that encode other sarcomeric proteins. Comprehensive open reading frame/splice site mutation analysis of TNNC1 performed on 1025 unrelated HCM patients enrolled over the last 10 years revealed novel missense mutations in TNNC1: A8V, C84Y, E134D, and D145E. Functional studies with these recombinant HcTnC HCM mutations showed increased Ca(2+) sensitivity of force development (A8V, C84Y and D145E) and force recovery (A8V and D145E). These results are consistent with the HCM functional phenotypes seen with other sarcomeric-HCM mutations (E134D showed no changes in these parameters). This is the largest cohort analysis of TNNC1 in HCM that details the discovery of at least three novel HCM-associated mutations and more strongly links TNNC1 to HCM along with functional evidence that supports a central role for its involvement in the disease. This study may help to further define TNNC1 as an HCM-susceptibility gene, a classification that has already been established for the other members of the troponin complex. Show less

In human cardiomyopathy, anatomical abnormalities such as hypertrophy and fibrosis contribute to the risk of ventricular arrhythmias and sudden death. Here we have shown that increased myofilament Ca2+ sensitivity, also a common feature in both inherited and acquired human cardiomyopathies, created arrhythmia susceptibility in mice, even in the absence of anatomical abnormalities. In mice expressing troponin T mutants that cause hypertrophic cardiomyopathy in humans, the risk of developing... Show moreIn human cardiomyopathy, anatomical abnormalities such as hypertrophy and fibrosis contribute to the risk of ventricular arrhythmias and sudden death. Here we have shown that increased myofilament Ca2+ sensitivity, also a common feature in both inherited and acquired human cardiomyopathies, created arrhythmia susceptibility in mice, even in the absence of anatomical abnormalities. In mice expressing troponin T mutants that cause hypertrophic cardiomyopathy in humans, the risk of developing ventricular tachycardia was directly proportional to the degree of Ca2+ sensitization caused by the troponin T mutation. Arrhythmia susceptibility was reproduced with the Ca2+-sensitizing agent EMD 57033 and prevented by myofilament Ca2+ desensitization with blebbistatin. Ca2+ sensitization markedly changed the shape of ventricular action potentials, resulting in shorter effective refractory periods, greater beat-to-beat variability of action potential durations, and increased dispersion of ventricular conduction velocities at fast heart rates. Together these effects created an arrhythmogenic substrate. Thus, myofilament Ca2+ sensitization represents a heretofore unrecognized arrhythmia mechanism. The protective effect of blebbistatin provides what we believe to be the first direct evidence that reduction of Ca2+ sensitivity in myofilaments is antiarrhythmic and might be beneficial to individuals with hypertrophic cardiomyopathy. Show less

Date Issued

2008

Identifier

FSU_migr_biomed_faculty_publications-0050, 10.1172/JCI36642

Format

Citation

Title

Mutagenesis of the Crystal Contact of Acidic Fibroblast Growth Factor.

An attempt has been made to improve a crystal contact of human acidic fibroblast growth factor (haFGF; 140 amino acids) to control the crystal growth, because haFGF crystallizes only as a thin-plate form, yielding crystals suitable for X-ray but not neutron diffraction. X-ray crystal analysis of haFGF showed that the Glu81 side chain, located at a crystal contact between haFGF molecules, is in close proximity with an identical residue related by crystallographic symmetry, suggesting that... Show moreAn attempt has been made to improve a crystal contact of human acidic fibroblast growth factor (haFGF; 140 amino acids) to control the crystal growth, because haFGF crystallizes only as a thin-plate form, yielding crystals suitable for X-ray but not neutron diffraction. X-ray crystal analysis of haFGF showed that the Glu81 side chain, located at a crystal contact between haFGF molecules, is in close proximity with an identical residue related by crystallographic symmetry, suggesting that charge repulsion may disrupt suitable crystal-packing interactions. To investigate whether the Glu residue affects the crystal-packing interactions, haFGF mutants in which Glu81 was replaced by Ala, Val, Leu, Ser and Thr were constructed. Although crystals of the Ala and Leu mutants were grown as a thin-plate form by the same precipitant (formate) as the wild type, crystals of the Ser and Thr mutants were grown with increased thickness, yielding a larger overall crystal volume. X-ray structural analysis of the Ser mutant determined at 1.35 A resolution revealed that the hydroxy groups of Ser are linked by hydrogen bonds mediated by the formate used as a precipitant. This approach to engineering crystal contacts may contribute to the development of large protein crystals for neutron crystallography. Show less

Date Issued

2008

Identifier

FSU_migr_biomed_faculty_publications-0007

Format

Citation

Title

S(1)' and S(2)' Subsite Specificities of Human Plasma Kallikrein and Tissue Kallikrein 1 for the Hydrolysis of Peptides Derived from the Bradykinin Domain of Human Kininogen.

The S(1)' and S(2)' subsite specificities of human tissue kallikrein 1 (KLK1) and human plasma kallikrein (HPK) were examined with the peptide series Abz-GFSPFRXSRIQ-EDDnp and Abz-GFSPFRSXRIQ-EDDnp [X=natural amino acids or S(PO(3)H(2))]. KLK1 efficiently hydrolyzed most of the peptides except those containing negatively charged amino acids at P(1)' and P(2)' positions. Abz-GFSPFRSSRIQ-EDDnp, as in human kininogen, is the best substrate for KLK1 and exclusively cleaved the R-S bond. All other... Show moreThe S(1)' and S(2)' subsite specificities of human tissue kallikrein 1 (KLK1) and human plasma kallikrein (HPK) were examined with the peptide series Abz-GFSPFRXSRIQ-EDDnp and Abz-GFSPFRSXRIQ-EDDnp [X=natural amino acids or S(PO(3)H(2))]. KLK1 efficiently hydrolyzed most of the peptides except those containing negatively charged amino acids at P(1)' and P(2)' positions. Abz-GFSPFRSSRIQ-EDDnp, as in human kininogen, is the best substrate for KLK1 and exclusively cleaved the R-S bond. All other peptides were cleaved also at the F-R bond. The synthetic human kininogen segment Abz-MISLMKRPPGFSPFRS(390)S(391)RI-NH(2) was hydrolyzed by KLK1 first at R-S and then at M-K bonds, releasing Lys-bradykinin. In the S(390) and S(391) phosphorylated analogs, this order of hydrolysis was inverted due to the higher resistance of the R-S bond. Abz-MISLMKRPPG-FSPFRSS(PO(3)H(2))(391)RI-NH(2) was hydrolyzed by KLK1 at M-K and mainly at the F-R bond, releasing des-(Arg(9))-Lys-Bk which is a B1 receptor agonist. HPK cleaved all the peptides at R and showed restricted specificity for S in the S(1)' subsite, with lower specificity for the S(2)' subsite. Abz-MISLMKRPPGFSPFRSSRI-NH(2) was efficiently hydrolyzed by HPK under bradykinin release, while the analogs containing S(PO(3)H(2)) were poorly hydrolyzed. In conclusion, S(1)' and S(2)' subsite specificities of KLK1 and HPK showed peculiarities that were observed with substrates containing the amino acid sequence of human kininogen. Show less

While protease-activated receptors (PARs) are known to mediate signaling events in CNS, contributing both to normal function and pathogenesis, the endogenous activators of CNS PARs are poorly characterized. In this study, we test the hypothesis that kallikreins (KLKs) represent an important pool of endogenous activators of CNS PARs. Specifically, KLK1 and KLK6 were examined for their ability to evoke intracellular Ca(2+) flux in a PAR-dependent fashion in NSC34 neurons and Neu7 astrocytes.... Show moreWhile protease-activated receptors (PARs) are known to mediate signaling events in CNS, contributing both to normal function and pathogenesis, the endogenous activators of CNS PARs are poorly characterized. In this study, we test the hypothesis that kallikreins (KLKs) represent an important pool of endogenous activators of CNS PARs. Specifically, KLK1 and KLK6 were examined for their ability to evoke intracellular Ca(2+) flux in a PAR-dependent fashion in NSC34 neurons and Neu7 astrocytes. Both KLKs were also examined for their ability to activate mitogen-activated protein kinases (extracellular signal-regulated kinases, C-Jun N-terminal kinases, and p38) and protein kinase B (AKT) intracellular signaling cascades. Cumulatively, these studies show that KLK6, but not KLK1, signals through PARs. KLK6 evoked intracellular Ca(2+) flux was mediated by PAR1 in neurons and both PAR1 and PAR2 in astrocytes. Importantly, both KLK1 and KLK6 altered the activation state of mitogen-activated protein kinases and AKT, suggestive of important roles for each in CNS neuron and glial differentiation, and survival. The cellular specificity of CNS-KLK activity was underscored by observations that both proteases promoted AKT activation in astrocytes, but inhibited such signaling in neurons. PAR1 and bradykinin receptor inhibitors were used to demonstrate that KLK1-mediated activation of extracellular signal-regulated kinases in neurons occurred in a non-PAR, bradykinin 2 (B2) receptor-dependent fashion, while similar signaling by KLK6 was mediated by the combined activation of PAR1 and B2. Cumulatively results indicate KLK6, but not KLK1 is an activator of CNS PARs, and that both KLKs are poised to signal in a B2 receptor-dependent fashion to regulate multiple signal transduction pathways relevant to CNS physiologic function and dysfunction. Show less

The human tissue kallikrein (KLK) family contains 15 secreted serine proteases that are expressed in a wide range of tissues and have been implicated in different physiological functions and disease states. Of these, KLK1 has been shown to be involved in the regulation of multiple physiological processes such as blood pressure, smooth muscle contraction, and vascular cell growth. KLK6 is overexpressed in breast and ovarian cancer tissues and has been shown to cleave peptide derived from human... Show moreThe human tissue kallikrein (KLK) family contains 15 secreted serine proteases that are expressed in a wide range of tissues and have been implicated in different physiological functions and disease states. Of these, KLK1 has been shown to be involved in the regulation of multiple physiological processes such as blood pressure, smooth muscle contraction, and vascular cell growth. KLK6 is overexpressed in breast and ovarian cancer tissues and has been shown to cleave peptide derived from human myelin protein and Abeta amyloid peptide in vitro. Here we analyzed the substrate specificity of KLK1 and KLK6, by substrate phage display using a random octapeptide library. Consistent with earlier biochemical data, KLK1 was shown to exhibit both trypsin- and chymotrypsin-like selectivities with Tyr/Arg preferred at site P1, Ser/Arg strongly preferred at P1', and Phe/Leu at P2. KLK6 displayed trypsin-like activity, with the P1 position occupied only by Arg and a strong preference for Ser in P1'. Docking simulations of consensus peptide provide information on the identity of the enzyme residues that are responsible for substrate binding. Bioinformatic analysis suggested several putative KLK6 protein substrates, such as ionotropic glutamate receptor (GluR) and synphilin. Show less

Date Issued

2008

Identifier

FSU_migr_biomed_faculty_publications-0006

Format

Citation

Title

Temporal control of the dephosphorylation of Cdk substrates by mitotic exit pathways in budding yeast.

The temporal phosphorylation of cell cycle-related proteins by cyclin-dependent kinases (Cdks) is critical for the correct order of cell cycle events. In budding yeast, CDC28 encodes the only Cdk and its association with various cyclins governs the temporal phosphorylation of Cdk substrates. S-phase Cdk substrates are phosphorylated earlier than mitotic Cdk substrates, which ensures the sequential order of DNA synthesis and mitosis. However, it remains unclear whether Cdk substrates are... Show moreThe temporal phosphorylation of cell cycle-related proteins by cyclin-dependent kinases (Cdks) is critical for the correct order of cell cycle events. In budding yeast, CDC28 encodes the only Cdk and its association with various cyclins governs the temporal phosphorylation of Cdk substrates. S-phase Cdk substrates are phosphorylated earlier than mitotic Cdk substrates, which ensures the sequential order of DNA synthesis and mitosis. However, it remains unclear whether Cdk substrates are dephosphorylated in temporally distinct windows. Cdc14 is a conserved protein phosphatase responsible for the dephosphorylation of Cdk substrates. In budding yeast, FEAR (Cdc14 early anaphase release) and MEN (mitotic exit network) activate phosphatase Cdc14 by promoting its release from the nucleolus in early and late anaphase, respectively. Here, we show that the sequential Cdc14 release and the distinct degradation timing of different cyclins provides the molecular basis for the differential dephosphorylation windows of S-phase and mitotic cyclin substrates. Our data also indicate that FEAR-induced dephosphorylation of S-phase Cdk substrates facilitates anaphase progression, revealing an extra layer of mitotic regulation. Show less

The mammalian molecular circadian clock in the suprachiasmatic nuclei (SCN) regulates locomotor activity rhythms as well as clocks in peripheral tissues (Reppert and Weaver, 2002; Ko and Takahashi, 2006). Constant light (LL) can induce behavioral and physiological arrhythmicity by desynchronizing clock cells in the SCN (Ohta et al., 2005). We examined how the disordered clock cells resynchronize by probing the molecular clock and measuring behavior in mice transferred from LL to constant... Show moreThe mammalian molecular circadian clock in the suprachiasmatic nuclei (SCN) regulates locomotor activity rhythms as well as clocks in peripheral tissues (Reppert and Weaver, 2002; Ko and Takahashi, 2006). Constant light (LL) can induce behavioral and physiological arrhythmicity by desynchronizing clock cells in the SCN (Ohta et al., 2005). We examined how the disordered clock cells resynchronize by probing the molecular clock and measuring behavior in mice transferred from LL to constant darkness (DD). The circadian locomotor activity rhythms disrupted in LL become robustly rhythmic again from the beginning of DD, and the starting phase of the rhythm in DD is specific, not random, suggesting that the desynchronized clock cells are quickly reset in an unconventional manner by the L/D transition. By measuring mPERIOD protein rhythms, we showed that the SCN and peripheral tissue clocks quickly become rhythmic again in phase with the behavioral rhythms. We propose that this resetting mechanism may be different from conventional phase shifting, which involves light induction of Period genes (Albrecht et al., 1997; Shearman et al., 1997; Shigeyoshi et al., 1997). Using our functional insights, we could shift the circadian phase of locomotor activity rhythms by 12 h using a 15 h LL treatment: essentially producing phase reversal by a single light pulse, a feat that has not been reported previously in wild-type mice and that has potential clinical utility. Show less

Tet 42, a novel tetracycline resistance determinant from deep subsurface bacteria, was characterized and found to have a 30% sequence similarity to TetA(Z). The protein is a putative efflux pump that shares characteristics with previously characterized pumps, including a divergently transcribed TetR repressor, a conserved GxxSDRxGRR motif, and transmembrane domains.

The human cardiac troponin I (hcTnI) mutation R145W has been associated with restrictive cardiomyopathy. In this study, simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145W transgenic mice (Tg-R145W) were explored. Tg-R145W fibers showed an approximately 13-16% increase in maximal Ca(2+)-activated force and ATPase activity compared to hcTnI wild-type transgenic mice. The force-generating cross-bridge turnover rate (g) and the energy cost (ATPase... Show moreThe human cardiac troponin I (hcTnI) mutation R145W has been associated with restrictive cardiomyopathy. In this study, simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145W transgenic mice (Tg-R145W) were explored. Tg-R145W fibers showed an approximately 13-16% increase in maximal Ca(2+)-activated force and ATPase activity compared to hcTnI wild-type transgenic mice. The force-generating cross-bridge turnover rate (g) and the energy cost (ATPase/force) were the same in all groups of fibers. Also, the Tg-R145W fibers showed a large increase in the Ca(2+) sensitivity of both force development and ATPase. In intact fibers, the mutation caused prolonged force and intracellular [Ca(2+)] transients and increased time to peak force. Analysis of force and Ca(2+) transients showed that there was a 40% increase in peak force in Tg-R145W muscles, which was likely due to the increased Ca(2+) transient duration. The above cited results suggest that: (1) there would be an increase in resistance to ventricular filling during diastole resulting from the prolonged force and Ca(2+) transients that would result in a decrease in ventricular filling (diastolic dysfunction); and (2) there would be a large (approximately 53%) increase in force during systole, which may help to partly compensate for diastolic dysfunction. These functional results help to explain the mechanisms by which these mutations give rise to a restrictive phenotype. Show less

Date Issued

2009

Identifier

FSU_migr_biomed_faculty_publications-0060, 10.1016/j.jmb.2009.07.080

Format

Citation

Title

Clinical and Functional Characterization of TNNT2 Mutations Identified in Patients with Dilated Cardiomyopathy.

BACKGROUND: A key issue for cardiovascular genetic medicine is ascertaining if a putative mutation indeed causes dilated cardiomyopathy (DCM). This is critically important as genetic DCM, usually presenting with advanced, life-threatening disease, may be preventable with early intervention in relatives known to carry the mutation. METHODS AND RESULTS: We recently undertook bidirectional resequencing of TNNT2, the cardiac troponin T gene, in 313 probands with DCM. We identified 6 TNNT2 protein... Show moreBACKGROUND: A key issue for cardiovascular genetic medicine is ascertaining if a putative mutation indeed causes dilated cardiomyopathy (DCM). This is critically important as genetic DCM, usually presenting with advanced, life-threatening disease, may be preventable with early intervention in relatives known to carry the mutation. METHODS AND RESULTS: We recently undertook bidirectional resequencing of TNNT2, the cardiac troponin T gene, in 313 probands with DCM. We identified 6 TNNT2 protein-altering variants in 9 probands, all who had early onset, aggressive disease. Additional family members of mutation carriers were then studied when available. Four of the 9 probands had DCM without a family history, and 5 probands had familial DCM. Only 1 mutation (Lys210del) could be attributed as definitively causative from previous reports. Four of the 5 missense mutations were novel (Arg134Gly, Arg151Cys, Arg159Gln, and Arg205Trp), and one was previously reported with hypertrophic cardiomyopathy (Glu244Asp). Based on the clinical, pedigree, and molecular genetic data, these 5 mutations were considered possibly or likely disease causing. To further clarify their potential pathophysiologic impact, we undertook functional studies of these mutations in cardiac myocytes reconstituted with mutant troponin T proteins. We observed decreased Ca(2+) sensitivity of force development, a hallmark of DCM, in support of the conclusion that these mutations are disease causing. CONCLUSIONS: We conclude that the combination of clinical, pedigree, molecular genetic, and functional data strengthen the interpretation of TNNT2 mutations in DCM. Show less

The rabbit is an important and de facto animal model in the study of ischemic disease and angiogenic therapy. Additionally, fibroblast growth factor 1 (FGF-1) is emerging as one of the most important growth factors for novel proangiogenic and pro-arteriogenic therapy. However, despite its significance, the fundamental biophysical properties of rabbit FGF-1, including its X-ray structure, have never been reported. Here, the cloning, crystallization, X-ray structure and determination of the... Show moreThe rabbit is an important and de facto animal model in the study of ischemic disease and angiogenic therapy. Additionally, fibroblast growth factor 1 (FGF-1) is emerging as one of the most important growth factors for novel proangiogenic and pro-arteriogenic therapy. However, despite its significance, the fundamental biophysical properties of rabbit FGF-1, including its X-ray structure, have never been reported. Here, the cloning, crystallization, X-ray structure and determination of the biophysical properties of rabbit FGF-1 are described. The X-ray structure shows that the amino-acid differences between human and rabbit FGF-1 are solvent-exposed and therefore potentially immunogenic, while the biophysical studies identify differences in thermostability and receptor-binding affinity that distinguish rabbit FGF-1 from human FGF-1. Show less

Large-volume protein crystals are a prerequisite for neutron diffraction studies and their production represents a bottleneck in obtaining neutron structures. Many protein crystals that permit the collection of high-resolution X-ray diffraction data are inappropriate for neutron diffraction owing to a plate-type morphology that limits the crystal volume. Human fibroblast growth factor 1 crystallizes in a plate morphology that yields atomic resolution X-ray diffraction data but has... Show moreLarge-volume protein crystals are a prerequisite for neutron diffraction studies and their production represents a bottleneck in obtaining neutron structures. Many protein crystals that permit the collection of high-resolution X-ray diffraction data are inappropriate for neutron diffraction owing to a plate-type morphology that limits the crystal volume. Human fibroblast growth factor 1 crystallizes in a plate morphology that yields atomic resolution X-ray diffraction data but has insufficient volume for neutron diffraction. The thin physical dimension has been identified as corresponding to the b cell edge and the X-ray structure identified a solvent-mediated crystal contact adjacent to position Glu81 that was hypothesized to limit efficient crystal growth in this dimension. In this report, a series of mutations at this crystal contact designed to both reduce side-chain entropy and replace the solvent-mediated interface with direct side-chain contacts are reported. The results suggest that improved crystal growth is achieved upon the introduction of direct crystal contacts, while little improvement is observed with side-chain entropy-reducing mutations alone. Show less

We previously reported the activation profiles of the human kallikrein-related peptidases (KLKs) as determined from a KLK pro-peptide fusion-protein system. That report described the activity profiles of 12 of the 15 mature KLKs versus the 15 different pro-KLK sequences. The missing profiles in the prior report, involving KLK9, 10, and 15, are now described. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, mass spectrometry, and N-terminal sequence analyses show that KLK9 and 10... Show moreWe previously reported the activation profiles of the human kallikrein-related peptidases (KLKs) as determined from a KLK pro-peptide fusion-protein system. That report described the activity profiles of 12 of the 15 mature KLKs versus the 15 different pro-KLK sequences. The missing profiles in the prior report, involving KLK9, 10, and 15, are now described. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, mass spectrometry, and N-terminal sequence analyses show that KLK9 and 10 exhibit low hydrolytic activities towards all of the 15 pro-KLK sequences, while KLK15 exhibits significant activity towards both Arg- and Lys-containing KLK pro-sequences. The ability of KLK15 to activate pro-KLK8, 12, and 14 is confirmed using recombinant pro-KLK proteins, and shown to be significant for activation of pro-KLK8 and 14, but not 12. These additional data for KLK9, 10, and 15 now permit a completed KLK activome profile, using a KLK pro-peptide fusion-protein system, to be described. The results suggest that KLK15, once activated, can potentially feed back into additional pro-KLK activation pathways. Conversely, KLK9 and 10, once activated, are unlikely to participate in further pro-KLK activation pathways, although similar to KLK1 they may activate other bioactive peptides. Show less

Date Issued

2009

Identifier

FSU_migr_biomed_faculty_publications-0012

Format

Citation

Title

Histone levels are regulated by phosphorylation and ubiquitylation-dependent proteolysis.

Histone levels are tightly regulated to prevent harmful effects such as genomic instability and hypersensitivity to DNA-damaging agents due to the accumulation of these highly basic proteins when DNA replication slows down or stops. Although chromosomal histones are stable, excess (non-chromatin bound) histones are rapidly degraded in a Rad53 (radiation sensitive 53) kinase-dependent manner in Saccharomyces cerevisiae. Here we demonstrate that excess histones associate with Rad53 in vivo and... Show moreHistone levels are tightly regulated to prevent harmful effects such as genomic instability and hypersensitivity to DNA-damaging agents due to the accumulation of these highly basic proteins when DNA replication slows down or stops. Although chromosomal histones are stable, excess (non-chromatin bound) histones are rapidly degraded in a Rad53 (radiation sensitive 53) kinase-dependent manner in Saccharomyces cerevisiae. Here we demonstrate that excess histones associate with Rad53 in vivo and seem to undergo modifications such as tyrosine phosphorylation and polyubiquitylation, before their proteolysis by the proteasome. We have identified the Tyr 99 residue of histone H3 as being critical for the efficient ubiquitylation and degradation of this histone. We have also identified the ubiquitin conjugating enzymes (E2) Ubc4 and Ubc5, as well as the ubiquitin ligase (E3) Tom1 (temperature dependent organization in mitotic nucleus 1), as enzymes involved in the ubiquitylation of excess histones. Regulated histone proteolysis has major implications for the maintenance of epigenetic marks on chromatin, genomic stability and the packaging of sperm DNA. Show less

The molecular function of the yeast polo-like kinase Cdc5 in Cdc14 release during early anaphase.

Creator

Liang, Fengshan, Jin, Fengzhi, Liu, Hong, Wang, Yanchang

Abstract/Description

In the budding yeast Saccharomyces cerevisiae, Cdc14 is sequestered within the nucleolus before anaphase entry through its association with Net1/Cfi1, a nucleolar protein. Protein phosphatase PP2A(Cdc55) dephosphorylates Net1 and keeps it as a hypophosphorylated form before anaphase. Activation of the Cdc fourteen early anaphase release (FEAR) pathway after anaphase entry induces a brief Cdc14 release from the nucleolus. Some of the components in the FEAR pathway, including Esp1, Slk19, and... Show moreIn the budding yeast Saccharomyces cerevisiae, Cdc14 is sequestered within the nucleolus before anaphase entry through its association with Net1/Cfi1, a nucleolar protein. Protein phosphatase PP2A(Cdc55) dephosphorylates Net1 and keeps it as a hypophosphorylated form before anaphase. Activation of the Cdc fourteen early anaphase release (FEAR) pathway after anaphase entry induces a brief Cdc14 release from the nucleolus. Some of the components in the FEAR pathway, including Esp1, Slk19, and Spo12, inactivate PP2A(Cdc55), allowing the phosphorylation of Net1 by mitotic cyclin-dependent kinase (Cdk) (Clb2-Cdk1). However, the function of another FEAR component, the Polo-like kinase Cdc5, remains elusive. Here, we show evidence indicating that Cdc5 promotes Cdc14 release primarily by stimulating the degradation of Swe1, the inhibitory kinase for mitotic Cdk. First, we found that deletion of SWE1 partially suppresses the FEAR defects in cdc5 mutants. In contrast, high levels of Swe1 impair FEAR activation. We also demonstrated that the accumulation of Swe1 in cdc5 mutants is responsible for the decreased Net1 phosphorylation. Therefore, we conclude that the down-regulation of Swe1 protein levels by Cdc5 promotes FEAR activation by relieving the inhibition on Clb2-Cdk1, the kinase for Net1 protein. Show less

Regulation of the transcription factor YY1 in mitosis through phosphorylation of its DNA-binding domain.

Creator

Rizkallah, Raed, Hurt, Myra M

Abstract/Description

Yin-Yang 1 (YY1) is a ubiquitously expressed zinc finger transcription factor. It regulates a vast array of genes playing critical roles in development, differentiation, and cell cycle. Very little is known about the mechanisms that regulate the functions of YY1. It has long been proposed that YY1 is a phosphoprotein; however, a direct link between phosphorylation and the function of YY1 has never been proven. Investigation of the localization of YY1 during mitosis shows that it is... Show moreYin-Yang 1 (YY1) is a ubiquitously expressed zinc finger transcription factor. It regulates a vast array of genes playing critical roles in development, differentiation, and cell cycle. Very little is known about the mechanisms that regulate the functions of YY1. It has long been proposed that YY1 is a phosphoprotein; however, a direct link between phosphorylation and the function of YY1 has never been proven. Investigation of the localization of YY1 during mitosis shows that it is distributed to the cytoplasm during prophase and remains excluded from DNA until early telophase. Immunostaining studies show that YY1 is distributed equally between daughter cells and rapidly associates with decondensing chromosomes in telophase, suggesting a role for YY1 in early marking of active and repressed genes. The exclusion of YY1 from DNA in prometaphase HeLa cells correlated with an increase in the phosphorylation of YY1 and loss of DNA-binding activity that can be reversed by dephosphorylation. We have mapped three phosphorylation sites on YY1 during mitosis and show that phosphorylation of two of these sites can abolish the DNA-binding activity of YY1. These results demonstrate a novel mechanism for the inactivation of YY1 through phosphorylation of its DNA-binding domain. Show less

Circadian rhythms in mammals are generated by a transcriptional negative feedback loop that is driven primarily by oscillations of PER and CRY, which inhibit their own transcriptional activators, CLOCK and BMAL1. Current models posit that CRY is the dominant repressor, while PER may play an accessory role. In this study, however, constitutive expression of PER, and not CRY1, severely disrupted the clock in fibroblasts and liver. Furthermore, constitutive expression of PER2 in the brain and... Show moreCircadian rhythms in mammals are generated by a transcriptional negative feedback loop that is driven primarily by oscillations of PER and CRY, which inhibit their own transcriptional activators, CLOCK and BMAL1. Current models posit that CRY is the dominant repressor, while PER may play an accessory role. In this study, however, constitutive expression of PER, and not CRY1, severely disrupted the clock in fibroblasts and liver. Furthermore, constitutive expression of PER2 in the brain and SCN of transgenic mice caused a complete loss of behavioral circadian rhythms in a conditional and reversible manner. These results demonstrate that rhythmic levels of PER2, rather than CRY1, are critical for circadian oscillations in cells and in the intact organism. Our biochemical evidence supports an elegant mechanism for the disparity: PER2 directly and rhythmically binds to CLOCK:BMAL1, while CRY only interacts indirectly; PER2 bridges CRY and CLOCK:BMAL1 to drive the circadian negative feedback loop. Show less

Circadian rhythms in mammals are generated by a negative transcriptional feedback loop in which PERIOD (PER) is rate-limiting for feedback inhibition. Casein kinases Idelta and Iepsilon (CKIdelta/epsilon) can regulate temporal abundance/activity of PER by phosphorylation-mediated degradation and cellular localization. Despite their potentially crucial effects on PER, it has not been demonstrated in a mammalian system that these kinases play essential roles in circadian rhythm generation as... Show moreCircadian rhythms in mammals are generated by a negative transcriptional feedback loop in which PERIOD (PER) is rate-limiting for feedback inhibition. Casein kinases Idelta and Iepsilon (CKIdelta/epsilon) can regulate temporal abundance/activity of PER by phosphorylation-mediated degradation and cellular localization. Despite their potentially crucial effects on PER, it has not been demonstrated in a mammalian system that these kinases play essential roles in circadian rhythm generation as does their homolog in Drosophila. To disrupt both CKIdelta/epsilon while avoiding the embryonic lethality of CKIdelta disruption in mice, we used CKIdelta-deficient Per2(Luc) mouse embryonic fibroblasts (MEFs) and overexpressed a dominant-negative mutant CKIepsilon (DN-CKIepsilon) in the mutant MEFs. CKIdelta-deficient MEFs exhibited a robust circadian rhythm, albeit with a longer period, suggesting that the cells possess a way to compensate for CKIdelta loss. When CKIepsilon activity was disrupted by the DN-CKIepsilon in the mutant MEFs, circadian bioluminescence rhythms were eliminated and rhythms in endogenous PER abundance and phosphorylation were severely compromised, demonstrating that CKIdelta/epsilon are indeed essential kinases for the clockwork. This is further supported by abolition of circadian rhythms when physical interaction between PER and CKIdelta/epsilon was disrupted by overexpressing the CKIdelta/epsilon binding domain of PER2 (CKBD-P2). Interestingly, CKBD-P2 overexpression led to dramatically low levels of endogenous PER, while PER-binding, kinase-inactive DN-CKIepsilon did not, suggesting that CKIdelta/epsilon may have a non-catalytic role in stabilizing PER. Our results show that an essential role of CKIdelta/epsilon is conserved between Drosophila and mammals, but CKIdelta/epsilon and DBT may have divergent non-catalytic functions in the clockwork as well. Show less

Mutations in TNNT2, encoding cardiac troponin T, commonly shows early onset, aggressive dilated cardiomyopathy (DCM). This observation may influence the decision of whether to undertake clinical genetic testing for TNNT2 in later onset DCM. Further, the trigger for late onset DCM remains enigmatic. A 70-year-old woman, previously healthy with a left ventricular ejection fraction of 50%-55% at age 69, presented with DCM of unknown cause and a 4-month history progressive heart failure requiring... Show moreMutations in TNNT2, encoding cardiac troponin T, commonly shows early onset, aggressive dilated cardiomyopathy (DCM). This observation may influence the decision of whether to undertake clinical genetic testing for TNNT2 in later onset DCM. Further, the trigger for late onset DCM remains enigmatic. A 70-year-old woman, previously healthy with a left ventricular ejection fraction of 50%-55% at age 69, presented with DCM of unknown cause and a 4-month history progressive heart failure requiring cardiac transplantation. Clinical genetic testing revealed a novel TNNT2 R139H mutation but no relevant variants in 18 other DCM genes. Her explanted heart showed partial fatty replacement in the right ventricle. Sequencing for five arrhythmogenic right ventricular dysplasia genes was negative. Functional studies in porcine cardiac skinned fibers reconstituted with the mutant R139H troponin T protein showed decreased Ca(2+) sensitivity at pH 7, characteristic of DCM. Because fatty infiltration may acidify the myocellular environment, maximal force development examined at pH 6.5 was diminished, suggesting a possible environmental trigger. We conclude that the TNNT2 R139H mutation was likely to be disease causing. Further, later age of onset may not be relevant to exclude genetic testing for TNNT2 mutations. Show less

A large body of emerging evidence indicates a functional interaction between the kallikrein-related peptidases (KLKs) and proteases of the thrombostasis axis. These interactions appear relevant for both normal health as well as pathologies associated with inflammation, tissue injury, and remodeling. Regulatory interactions between the KLKs and thrombostasis proteases could impact several serious human diseases, including neurodegeneration and cancer. The emerging network of specific... Show moreA large body of emerging evidence indicates a functional interaction between the kallikrein-related peptidases (KLKs) and proteases of the thrombostasis axis. These interactions appear relevant for both normal health as well as pathologies associated with inflammation, tissue injury, and remodeling. Regulatory interactions between the KLKs and thrombostasis proteases could impact several serious human diseases, including neurodegeneration and cancer. The emerging network of specific interactions between these two protease families appears to be complex, and much work remains to elucidate it. Complete understanding how this functional network resolves over time, given specific initial conditions, and how it might be controllably manipulated, will probably contribute to the emergence of novel diagnostics and therapeutic agents for major diseases. Show less

Given both the high prevalence of anxiety disorders in women and the fact that little is known about the mechanisms of gender differences in anxiety, our primary aim in this study was to investigate the neurobiological mechanisms underlying sex differences in social anxiety-like behavior in rats. Through the use of zif268 antisense oligodeoxynucleotides (zif ASO), we induced a temporary downregulation of zif268 expression in the medial prefrontal cortex of male and female rats and found that... Show moreGiven both the high prevalence of anxiety disorders in women and the fact that little is known about the mechanisms of gender differences in anxiety, our primary aim in this study was to investigate the neurobiological mechanisms underlying sex differences in social anxiety-like behavior in rats. Through the use of zif268 antisense oligodeoxynucleotides (zif ASO), we induced a temporary downregulation of zif268 expression in the medial prefrontal cortex of male and female rats and found that zif268 ASO male rats show more social anxiety-like behaviors when compared with control male rats in the social interaction test. In fact, zif268 ASO males displayed social anxiety-like behaviors, which were similar to control females, thus downregulation of zif268 expression in the mPFC of male rats eliminated sex differences previously found in the social anxiety-like behavior tests. Interestingly, zif268 ASO in female rats had no effect on their social interaction. Our novel findings have led us to ascertain that sexually dimorphic zif268 expression in the mPFC is a key molecular factor in mediating sex-specific anxiety-like behavior in the social interaction test. Show less

Mutations in sarcomeric proteins have recently been established as heritable causes of Restrictive Cardiomyopathy (RCM). RCM is clinically characterized as a defect in cardiac diastolic function, such as, impaired ventricular relaxation, reduced diastolic volume and increased end-diastolic pressure. To date, mutations have been identified in the cardiac genes for desmin, alpha-actin, troponin I and troponin T. Functional studies in skinned muscle fibers reconstituted with troponin mutants... Show moreMutations in sarcomeric proteins have recently been established as heritable causes of Restrictive Cardiomyopathy (RCM). RCM is clinically characterized as a defect in cardiac diastolic function, such as, impaired ventricular relaxation, reduced diastolic volume and increased end-diastolic pressure. To date, mutations have been identified in the cardiac genes for desmin, alpha-actin, troponin I and troponin T. Functional studies in skinned muscle fibers reconstituted with troponin mutants have established phenotypes consistent with the clinical findings which include an increase in myofilament Ca(2+) sensitivity and basal force. Moreover, when RCM mutants are incorporated into reconstituted myofilaments, the ability to inhibit the ATPase activity is reduced. A majority of the mutations cluster in specific regions of cardiac troponin and appear to be mutational "hot spots". This paper highlights the functional and clinical characteristics of RCM linked mutations within the troponin complex. Show less

Date Issued

2010

Identifier

FSU_migr_biomed_faculty_publications-0053, 10.1155/2010/350706

Format

Citation

Title

THE ROLE OF INTRACELLULAR SODIUM (Na) IN THE REGULATION OF CALCIUM (Ca)-MEDIATED SIGNALING AND TOXICITY.

Creator

Yu, Xian-Min, Groveman, Bradley R, Fang, Xiao-Qian, Lin, Shuang-Xiu

Abstract/Description

It is known that activated N-methyl-D-aspartate receptors (NMDARs) are a major route of excessive calcium ion (Ca(2+)) entry in central neurons, which may activate degradative processes and thereby cause cell death. Therefore, NMDARs are now recognized to play a key role in the development of many diseases associated with injuries to the central nervous system (CNS). However, it remains a mystery how NMDAR activity is recruited in the cellular processes leading to excitotoxicity and how NMDAR... Show moreIt is known that activated N-methyl-D-aspartate receptors (NMDARs) are a major route of excessive calcium ion (Ca(2+)) entry in central neurons, which may activate degradative processes and thereby cause cell death. Therefore, NMDARs are now recognized to play a key role in the development of many diseases associated with injuries to the central nervous system (CNS). However, it remains a mystery how NMDAR activity is recruited in the cellular processes leading to excitotoxicity and how NMDAR activity can be controlled at a physiological level. The sodium ion (Na(+)) is the major cation in extracellular space. With its entry into the cell, Na(+) can act as a critical intracellular second messenger that regulates many cellular functions. Recent data have shown that intracellular Na(+) can be an important signaling factor underlying the up-regulation of NMDARs. While Ca(2+) influx during the activation of NMDARs down-regulates NMDAR activity, Na(+) influx provides an essential positive feedback mechanism to overcome Ca(2+)-induced inhibition and thereby potentiate both NMDAR activity and inward Ca(2+) flow. Extensive investigations have been conducted to clarify mechanisms underlying Ca(2+)-mediated signaling. This review focuses on the roles of Na(+) in the regulation of Ca(2+)-mediated NMDAR signaling and toxicity. Show less

Although the Notch signaling pathway is one of the most intensely studied intracellular signaling pathways, the mechanisms by which Notch signaling regulates transcription remain incompletely understood. Here, we report that B cell leukemia/lymphoma 6 (BCL6), a transcriptional repressor, is a Notch-associated factor. BCL6 is necessary to maintain the expression of Pitx2 in the left lateral plate mesoderm during the patterning of left-right asymmetry in Xenopus embryos. For this process, BCL6... Show moreAlthough the Notch signaling pathway is one of the most intensely studied intracellular signaling pathways, the mechanisms by which Notch signaling regulates transcription remain incompletely understood. Here, we report that B cell leukemia/lymphoma 6 (BCL6), a transcriptional repressor, is a Notch-associated factor. BCL6 is necessary to maintain the expression of Pitx2 in the left lateral plate mesoderm during the patterning of left-right asymmetry in Xenopus embryos. For this process, BCL6 forms a complex with BCL6 corepressor (BCoR) on the promoters of selected Notch target genes such as enhancer of split related 1. BCL6 also inhibits the transcription of these genes by competing for the Notch1 intracellular domain, preventing the coactivator Mastermind-like1 (MAM1) from binding. These results define a mechanism restricting Notch-activated transcription to cell-type-appropriate subsets of target genes, and elucidate its relevance in vivo during left-right asymmetric development. Show less

Ovariectomy (OVX) leads to hyperphagia and weight gain in rats, which can be prevented by estradiol (E2) replacement; however, the role of endogenous E2 on feeding and energy homeostasis in female mice has not been well characterized. The primary goal of this study was to assess the relative contribution of increased energy intake and decreased energy expenditure to OVX-induced weight gain in female rats and mice. OVX led to hyperphagia in rats, but did not produce daily, nor cumulative,... Show moreOvariectomy (OVX) leads to hyperphagia and weight gain in rats, which can be prevented by estradiol (E2) replacement; however, the role of endogenous E2 on feeding and energy homeostasis in female mice has not been well characterized. The primary goal of this study was to assess the relative contribution of increased energy intake and decreased energy expenditure to OVX-induced weight gain in female rats and mice. OVX led to hyperphagia in rats, but did not produce daily, nor cumulative, hyperphagia in mice. OVX decreased mass-specific metabolic rate in mice, but not in rats. OVX decreased home cage locomotor activity in both species. Pair-feeding attenuated OVX-induced weight gain in rats and produced both short- and long-term changes in expression of key hypothalamic genes involved in food intake and energy homeostasis, i.e., the anorexigenic neuropeptide pro-opiomelanocortin (POMC) and the orexigenic neuropeptides: melanin-concentrating hormone (MCH) and agouti-related peptide (AgRP). No differences in hypothalamic gene expression were observed between OVX'd and sham mice. The results suggest that OVX-induced weight gain is mediated by hyperphagia and reduced locomotor activity in rats, but that in mice, it is primarily mediated by reduced locomotor activity and metabolic rate. Show less

Studies have shown that labor occurs primarily in the night/morning hours. Recently, we identified the human myometrium as a target for melatonin (MEL), the neuroendocrine output signal coding for circadian night. The purpose of this study was to determine the signaling pathway underlying the effects of MEL on contractility and the contractile machinery in immortalized human myometrial cells. To ascertain the signaling pathway of MEL leading to its effects on myometrial contractility in vitro... Show moreStudies have shown that labor occurs primarily in the night/morning hours. Recently, we identified the human myometrium as a target for melatonin (MEL), the neuroendocrine output signal coding for circadian night. The purpose of this study was to determine the signaling pathway underlying the effects of MEL on contractility and the contractile machinery in immortalized human myometrial cells. To ascertain the signaling pathway of MEL leading to its effects on myometrial contractility in vitro, we performed gel retraction assays with cells exposed to iodo-MEL (I-MEL) with or without oxytocin and the Rho kinase inhibitor Y27632. I-MEL effects on inositol trisphosphate (IP(3))/diacylglycerol (DAG)/protein kinase C (PKC) signaling were also investigated. Additionally, we assayed for caldesmon phosphorylation and ERK1/2 activation. I-MEL was found to activate PKC alpha via the phospholipase C/IP(3)/DAG signaling pathway, which was confirmed by PKC enzyme assay. I-MEL did not affect myosin light chain phosphatase activity, and its effects on contractility were insensitive to Rho kinase inhibition. I-MEL did increase phosphorylation of ERK1/2 and caldesmon, which was inhibited by the MAPK kinase inhibitor PD98059 or the PKC inhibitor C1. MEL sensitizes myometrial cells to subsequent procontractile signals in vitro through activation of the phospholipase C/IP(3)/DAG signaling pathway, resulting in specific activation of PKC alpha and ERK1/2, thereby phosphorylating caldesmon, which increases actin availability for myosin binding and cross-bridging. In vivo, this sensitization would provide a mechanism for the increased nocturnal uterine contractility and labor that has been observed in late-term human pregnancy. Show less

Intracellular linkers are involved in Mg2+-dependent modulation of the Eag potassium channel.

Creator

Liu, Xinqiu, Wu, Yuying, Zhou, Yi

Abstract/Description

Modulation of activation kinetics by divalent ions is one of the characteristic features of Eag channels. Here, we report that Mg(2+)-dependent deceleration of Eag channel activation is significantly attenuated by a G297E mutation, which exhibits a gain-of-function phenotype in Drosophila by suppressing the effect of shaker mutation on behavior and neuronal excitability. The G297 residue is located in the intracellular linker of transmembrane segments S2 and S3, and is thus not involved in... Show moreModulation of activation kinetics by divalent ions is one of the characteristic features of Eag channels. Here, we report that Mg(2+)-dependent deceleration of Eag channel activation is significantly attenuated by a G297E mutation, which exhibits a gain-of-function phenotype in Drosophila by suppressing the effect of shaker mutation on behavior and neuronal excitability. The G297 residue is located in the intracellular linker of transmembrane segments S2 and S3, and is thus not involved in direct binding of Mg(2+) ions. Moreover, mutation of the only positively charged residue in the other intracellular linker between S4 and S5 also results in a dramatic reduction of Mg(2+)-dependent modulation of Eag activation kinetics. Collectively, the two mutations in eag eliminate or even paradoxically reverse the effect of Mg(2+) on channel activation and inactivation kinetics. Together, these results suggest an important role of the intracellular linker regions in gating processes of Eag channels. Show less

Dosage compensation equates between the sexes the gene dose of sex chromosomes that carry substantially different gene content. In Drosophila, the single male X chromosome is hypertranscribed by approximately two-fold to effect this correction. The key genes are male lethal and appear not to be required in females, or affect their viability. Here, we show these male lethals do in fact have a role in females, and they participate in the very process which will eventually shut down their... Show moreDosage compensation equates between the sexes the gene dose of sex chromosomes that carry substantially different gene content. In Drosophila, the single male X chromosome is hypertranscribed by approximately two-fold to effect this correction. The key genes are male lethal and appear not to be required in females, or affect their viability. Here, we show these male lethals do in fact have a role in females, and they participate in the very process which will eventually shut down their function--female determination. We find the male dosage compensation complex is required for upregulating transcription of the sex determination master switch, Sex-lethal, an X-linked gene which is specifically activated in females in response to their two X chromosomes. The levels of some X-linked genes are also affected, and some of these genes are used in the process of counting the number of X chromosomes early in development. Our data suggest that before the female state is set, the ground state is male and female X chromosome expression is elevated. Females thus utilize the male dosage compensation process to amplify the signal which determines their fate. Show less

Type I collagen, synthesized in all tissues as the heterotrimer of two alpha1(I) polypeptides and one alpha2(I) polypeptide, is the most abundant protein in the human body. Here we show that intact nonmuscle myosin filaments are required for the synthesis of heterotrimeric type I collagen. Conserved 5' stem-loop in collagen alpha1(I) and alpha2(I) mRNAs binds the RNA-binding protein LARP6. LARP6 interacts with nonmuscle myosin through its C-terminal domain and associates collagen mRNAs with... Show moreType I collagen, synthesized in all tissues as the heterotrimer of two alpha1(I) polypeptides and one alpha2(I) polypeptide, is the most abundant protein in the human body. Here we show that intact nonmuscle myosin filaments are required for the synthesis of heterotrimeric type I collagen. Conserved 5' stem-loop in collagen alpha1(I) and alpha2(I) mRNAs binds the RNA-binding protein LARP6. LARP6 interacts with nonmuscle myosin through its C-terminal domain and associates collagen mRNAs with the filaments. Dissociation of nonmuscle myosin filaments results in secretion of collagen alpha1(I) homotrimer, diminished intracellular colocalization of collagen alpha1(I) and alpha2(I) polypeptides (required for folding of the heterotrimer), and their increased intracellular degradation. Inhibition of the motor function of myosin has similar collagen-specific effects, while disruption of actin filaments has a general effect on protein secretion. Nonmuscle myosin copurifies with polysomes, and there is a subset of polysomes involved in myosin-dependent translation of collagen mRNAs. These results indicate that association of collagen mRNAs with nonmuscle myosin filaments is necessary to coordinately synthesize collagen alpha1(I) and alpha2(I) polypeptides. We postulate that LARP6/myosin-dependent mechanism regulates the synthesis of heterotrimeric type I collagen by coordinating the translation of collagen mRNAs. Show less

Neuronal Src (n-Src) is an alternative isoform of Src kinase containing a 6-amino acid insert in the SH3 domain that is highly expressed in neurons of the central nervous system (CNS). To investigate the function of n-Src, wild-type n-Src, constitutively active n-Src in which the C-tail tyrosine 535 was mutated to phenylalanine (n-Src/Y535F) and inactive n-Src in which the lysine 303 was mutated to arginine in addition to the mutation of Y535F (n-Src/K303R/Y535F), were expressed and purified... Show moreNeuronal Src (n-Src) is an alternative isoform of Src kinase containing a 6-amino acid insert in the SH3 domain that is highly expressed in neurons of the central nervous system (CNS). To investigate the function of n-Src, wild-type n-Src, constitutively active n-Src in which the C-tail tyrosine 535 was mutated to phenylalanine (n-Src/Y535F) and inactive n-Src in which the lysine 303 was mutated to arginine in addition to the mutation of Y535F (n-Src/K303R/Y535F), were expressed and purified from Escherichia coli BL21(DE3) cells. We found that all three types of n-Src constructs expressed at very high yields (∼500 mg/L) at 37°C, but formed inclusion bodies. In the presence of 8M urea these proteins could be solubilized, purified under denaturing conditions, and subsequently refolded in the presence of arginine (0.5M). These Src proteins were enzymatically active except for the n-Src/K303R/Y535F mutant. n-Src proteins expressed at 18°C were soluble, albeit at lower yields (∼10-20 mg/L). The lowest yields were for n-Src/Y535F (∼10 mg/L) and the highest for n-Src/K303R/Y535F (∼20 mg/L). We characterized the purified n-Src proteins expressed at 18°C. We found that altering n-Src enzyme activity either pharmacologically (e.g., application of ATP or a Src inhibitor) or genetically (mutation of Y535 or K303) was consistently associated with changes in n-Src stability: an increase in n-Src activity was coupled with a decrease in n-Src stability and vice versa. These findings, therefore, indicate that n-Src activity and stability are interdependent. Finally, the successful production of functionally active n-Src in this study indicates that the bacterial expression system may be a useful protein source in future investigations of n-Src regulation and function. Show less

Cutaneous malignant melanoma is an aggressive disease of poor prognosis. Clinical and experimental studies have provided major insight into the pathogenesis of the disease, including the functional interaction between melanoma cells and surrounding keratinocytes, fibroblasts, and immune cells. Nevertheless, patients with metastasized melanoma have a very poor prognosis and are largely refractory to clinical therapies. Hence, diagnostic tools to monitor melanoma development, as well as... Show moreCutaneous malignant melanoma is an aggressive disease of poor prognosis. Clinical and experimental studies have provided major insight into the pathogenesis of the disease, including the functional interaction between melanoma cells and surrounding keratinocytes, fibroblasts, and immune cells. Nevertheless, patients with metastasized melanoma have a very poor prognosis and are largely refractory to clinical therapies. Hence, diagnostic tools to monitor melanoma development, as well as therapeutic targets, are urgently needed. We investigated the expression pattern of the kallikrein-related peptidase 6 (KLK6) in human melanoma tissue sections throughout tumor development. Although KLK6 was not detectable in tumor cells, we found strong KLK6 protein expression in keratinocytes and stromal cells located adjacent to benign nevi, primary melanomas, and cutaneous metastatic lesions, suggesting a paracrine function of extracellular KLK6 during neoplastic transformation and malignant progression. Accordingly, recombinant Klk6 protein significantly induced melanoma cell migration and invasion accompanied by an accelerated intracellular Ca(2+) flux. We could further demonstrate that KLK6-induced intracellular Ca(2+) flux and tumor cell invasion critically depends on the protease-activated receptor 1 (PAR1). Our data provide experimental evidence that specific inhibition of the KLK6-PAR1 axis may interfere with the deleterious effect of tumor-microenvironment interaction and represent a potential option for translational melanoma research. Show less

BACKGROUND: Kallikrein 6 (KLK6) is a newly identified member of the kallikrein family of secreted serine proteases that prior studies indicate is elevated at sites of central nervous system (CNS) inflammation and which shows regulated expression with T cell activation. Notably, KLK6 is also elevated in the serum of multiple sclerosis (MS) patients however its potential roles in immune function are unknown. Herein we specifically examine whether KLK6 alters immune cell survival and the... Show moreBACKGROUND: Kallikrein 6 (KLK6) is a newly identified member of the kallikrein family of secreted serine proteases that prior studies indicate is elevated at sites of central nervous system (CNS) inflammation and which shows regulated expression with T cell activation. Notably, KLK6 is also elevated in the serum of multiple sclerosis (MS) patients however its potential roles in immune function are unknown. Herein we specifically examine whether KLK6 alters immune cell survival and the possible mechanism by which this may occur. METHODOLOGY/PRINCIPAL FINDINGS: Using murine whole splenocyte preparations and the human Jurkat T cell line we demonstrate that KLK6 robustly supports cell survival across a range of cell death paradigms. Recombinant KLK6 was shown to significantly reduce cell death under resting conditions and in response to camptothecin, dexamethasone, staurosporine and Fas-ligand. Moreover, KLK6-over expression in Jurkat T cells was shown to generate parallel pro-survival effects. In mixed splenocyte populations the vigorous immune cell survival promoting effects of KLK6 were shown to include both T and B lymphocytes, to occur with as little as 5 minutes of treatment, and to involve up regulation of the pro-survival protein B-cell lymphoma-extra large (Bcl-XL), and inhibition of the pro-apoptotic protein Bcl-2-interacting mediator of cell death (Bim). The ability of KLK6 to promote survival of splenic T cells was also shown to be absent in cell preparations derived from PAR1 deficient mice. CONCLUSION/SIGNIFICANCE: KLK6 promotes lymphocyte survival by a mechanism that depends in part on activation of PAR1. These findings point to a novel molecular mechanism regulating lymphocyte survival that is likely to have relevance to a range of immunological responses that depend on apoptosis for immune clearance and maintenance of homeostasis. Show less

Distribution of orientations of myosin was examined in ex-vivo myofibrils from hearts of transgenic (Tg) mice expressing Familial Hypertrophic Cardiomyopathy (FHC) troponin T (TnT) mutations I79N, F110I and R278C. Humans are heterozygous for sarcomeric FHC mutations and so hypertrophic myocardium contains a mixture of the wild-type (WT) and mutated (MUT) TnT. If mutations are expressed at a low level there may not be a significant change in the global properties of heart muscle. In contrast,... Show moreDistribution of orientations of myosin was examined in ex-vivo myofibrils from hearts of transgenic (Tg) mice expressing Familial Hypertrophic Cardiomyopathy (FHC) troponin T (TnT) mutations I79N, F110I and R278C. Humans are heterozygous for sarcomeric FHC mutations and so hypertrophic myocardium contains a mixture of the wild-type (WT) and mutated (MUT) TnT. If mutations are expressed at a low level there may not be a significant change in the global properties of heart muscle. In contrast, measurements from a few molecules avoid averaging inherent in the global measurements. It is thus important to examine the properties of only a few molecules of muscle. To this end, the lever arm of one out of every 60,000 myosin molecules was labeled with a fluorescent dye and a small volume within the A-band (~1 fL) was observed by confocal microscopy. This volume contained on average 5 fluorescent myosin molecules. The lever arm assumes different orientations reflecting different stages of acto-myosin enzymatic cycle. We measured the distribution of these orientations by recording polarization of fluorescent light emitted by myosin-bound fluorophore during rigor and contraction. The distribution of orientations of rigor WT and MUT myofibrils was significantly different. There was a large difference in the width and of skewness and kurtosis of rigor distributions. These findings suggest that the hypertrophic phenotype associated with the TnT mutations can be characterized by a significant increase in disorder of rigor cross-bridges. Show less

Experimental Support for the Evolution of Symmetric Protein Architecture from a Simple Peptide Motif.

Creator

Lee, Jihun, Blaber, Michael

Abstract/Description

The majority of protein architectures exhibit elements of structural symmetry, and "gene duplication and fusion" is the evolutionary mechanism generally hypothesized to be responsible for their emergence from simple peptide motifs. Despite the central importance of the gene duplication and fusion hypothesis, experimental support for a plausible evolutionary pathway for a specific protein architecture has yet to be effectively demonstrated. To address this question, a unique "top-down... Show moreThe majority of protein architectures exhibit elements of structural symmetry, and "gene duplication and fusion" is the evolutionary mechanism generally hypothesized to be responsible for their emergence from simple peptide motifs. Despite the central importance of the gene duplication and fusion hypothesis, experimental support for a plausible evolutionary pathway for a specific protein architecture has yet to be effectively demonstrated. To address this question, a unique "top-down symmetric deconstruction" strategy was utilized to successfully identify a simple peptide motif capable of recapitulating, via gene duplication and fusion processes, a symmetric protein architecture (the threefold symmetric β-trefoil fold). The folding properties of intermediary forms in this deconstruction agree precisely with a previously proposed "conserved architecture" model for symmetric protein evolution. Furthermore, a route through foldable sequence-space between the simple peptide motif and extant protein fold is demonstrated. These results provide compelling experimental support for a plausible evolutionary pathway of symmetric protein architecture via gene duplication and fusion processes. Show less